The phospholipid Platelet-activating factor (PAF) is perhaps the most powerful pro-inflammatory and prothromobtic[unreadable] lipid mediator yet defined. All cellular components of the acute inflammatory system express the[unreadable] single known receptor for PAF. PAF is synthesized by, and retained on the surface of, activated endothelial[unreadable] ceils where it stimulates tethered leukocytes. PAF accumulation normally is tightly controlled, but PAF has a[unreadable] role in thrombosis, sepsis and reperfusion damage.[unreadable] There are deficits in our knowledge of how, when, and where PAF receptor ligands are formed, how PAF is[unreadable] presented to cells of the innate immune system and how PAF stimulated cells interact with other cells. We[unreadable] do not know whether PAF receptor ligands are formed during stent placement, whether PAF stimulated[unreadable] leukocytes provide platelets with substrate for thromboxane production even when platelet cyclooxygenase[unreadable] has been inhibited by aspirin, and we do not know whether PAF production by leukocytes stimulated with[unreadable] endotoxin is mimicked by endogenous agonists that include anti-phospholipid antibodies.[unreadable] We propose to identify the pathways leading to activation of the rate-limiting PAF synthetic activity,[unreadable] determine whether individuals vary in their ability to make PAF, take advantage of new information and[unreadable] techniques to purify the enzyme responsible for PAF synthesis for eventual rational inhibitor design, and[unreadable] identify and modulate mechanisms that cause microparticle formation and PAF release, and to determine[unreadable] whether these parameters correlate with thrombosis. We will define the pro-thrombotic lipids released during[unreadable] stent placement, we will investigate alternate routes to generate thromboxane Aa, and determine whether this[unreadable] correlates with susceptibility to slow reflow after stent placement, and we will test alternate, endogenous[unreadable] ligands of TLR4 as leukocyte agonists. We have four aims:[unreadable] Aim 1. Mechanistically define adhesion-dependent PAF synthesis in PMN.[unreadable] Aim 2. Purify and molecularly characterize the leukocyte PAF acetyltransferase synthetic enzyme.[unreadable] Aim 3. Identify factors that extend the effect of PAF through microparticle release.[unreadable] Aim 4. Define novel routes to leukocyte activation and inflammatory mediator production[unreadable]